CN105505383A - A kind of synthesis method of fluorescent copper nanocluster - Google Patents

Abstract

The invention relates to a synthesis method of a fluorescent copper nanocluster, belonging to the technical field of biochemistry²⁺Uniformly mixing the solution and the bovine serum albumin solution, adding a reducing agent, adjusting the pH value to 10-13, and standing for 6-30 hours; the Cu²⁺And the molar ratio of the bovine serum albumin to the reducing agent is 1: 4: 1-320; the reducing agent is at least one of hydroxylamine hydrochloride, L-ascorbic acid, D-glucose, sodium borohydride and sodium citrate, and the method has the advantages that the synthesized fluorescent copper nano cluster has bright emitted fluorescence, small size, high stability, good water solubility and the like.

Description

A kind of synthesis method of fluorescent copper nanocluster

technical field

The invention relates to a method for synthesizing fluorescent copper nanoclusters, belonging to the technical field of biochemistry.

Background technique

In recent years, ultrasmall fluorescent metal nanoclusters containing a few to tens of metal atoms, linking atoms and large-sized nanoparticles, have attracted extensive attention due to their size-dependent unique physical and chemical properties. Since the size of metal nanoclusters is similar to the Fermi wavelength of electrons, it exhibits discrete energy levels and molecular-like properties, such as size-tunable electronic transition states and strong fluorescence properties. Metal nanoclusters are considered as novel, potential, and practical nanomaterials with practical applications in many fields, such as: cell imaging, ion sensing, and catalysis. Compared with traditional fluorescent quantum dots, metal nanoclusters have many superior properties, such as: low toxicity, ultra-small size, good biocompatibility and multifunctional surface chemical properties, which make them more and more popular in the field of biology. Much attention.

In the past ten years, extensive research on fluorescent metal nanoclusters has mainly focused on the synthesis of fluorescent Au, Ag, and Pt nanoclusters, applying various types of templates, such as: peptides, proteins, DNA, polymers, dendrimers Polymers and biothiol small molecules. However, Au, Ag, and Pt are noble metals, and thus, the synthesis of Au, Ag, and Pt nanoclusters is very expensive. As we all know, Cu, a non-precious metal element, is abundant on the earth, and it is cheap and easy to get in daily life. As a potential nanomaterial, copper nanoclusters can be applied in many research fields such as catalysis, sensing, cell labeling and cell imaging. However, since the synthesis of ultra-small sized copper nanoclusters is very difficult, and even the synthesized copper nanoclusters are easily oxidized in air, the preparation of ultra-small sized and highly stable copper nanoclusters is still a major challenge at present. , therefore, the synthesis of Cu nanoclusters is still at a preliminary stage compared with the synthetic methods of Au and Ag nanoclusters.

Contents of the invention

The invention synthesizes fluorescent copper nano-clusters with bright emission fluorescence, good water solubility, high stability, good biocompatibility and low toxicity by adding a new reducing agent system.

The invention provides a method for synthesizing fluorescent copper nanoclusters. The synthesis method is to mix the solution containing Cu ²⁺ with the bovine serum albumin solution, then add a reducing agent, adjust the pH to 10-13, and stand for 6 ~30h;

The molar ratio of Cu ²⁺ , bovine serum albumin and reducing agent is 1:4:1-320;

The reducing agent is at least one of hydroxylamine hydrochloride, L-ascorbic acid, D-glucose, sodium borohydride and sodium citrate.

The reducing agent of the present invention is preferably one of hydroxylamine hydrochloride, L-ascorbic acid, D-glucose, sodium borohydride and sodium citrate.

The reducing agent in the present invention is preferably hydroxylamine hydrochloride, and the molar ratio of Cu ²⁺ , bovine serum albumin and hydroxylamine hydrochloride is more preferably 1:4:40-240.

The reducing agent in the present invention is preferably L-ascorbic acid, and the molar ratio of Cu ²⁺ , bovine serum albumin and L-ascorbic acid is more preferably 1:4:2-60.

The reducing agent in the present invention is preferably D-glucose, and the molar ratio of Cu ²⁺ , bovine serum albumin and D-glucose is more preferably 1:4:20-160.

The reducing agent in the present invention is preferably sodium borohydride, and the molar ratio of Cu ²⁺ , bovine serum albumin and sodium borohydride is more preferably 1:4:4-8.

The reducing agent in the present invention is preferably sodium citrate, and the molar ratio of Cu ²⁺ , bovine serum albumin and sodium citrate is more preferably 1:4:40-320.

The reducing agent of the present invention is preferably a co-reductant system of sodium borohydride and sodium citrate, or a co-reductant system of sodium borohydride and D-glucose, or a co-reductant system of sodium borohydride and L-ascorbic acid, or D-glucose and L-ascorbic acid co-reductant system, or D-glucose and hydroxylamine hydrochloride co-reductant system, or L-ascorbic acid and hydroxylamine hydrochloride co-reductant system.

The reducing agent in the present invention is preferably a co-reducing agent system of sodium borohydride and sodium citrate, and the molar ratio of Cu ²⁺ , bovine serum albumin, sodium borohydride and sodium citrate is more preferably 1:4:1-8 : 20-160.

The reducing agent in the present invention is preferably a co-reducing agent system of sodium borohydride and D-glucose, and the molar ratio of Cu ²⁺ , bovine serum albumin, sodium borohydride and D-glucose is more preferably 1:4:1-8 : 20-160.

The reducing agent of the present invention is preferably a co-reducing agent system of sodium borohydride and L-ascorbic acid, and the molar ratio of Cu ²⁺ , bovine serum albumin, sodium borohydride and L-ascorbic acid is more preferably 1:4:1-8 : 20-160.

The reducing agent in the present invention is preferably D-glucose and L-ascorbic acid co-reducing agent system. The molar ratio of Cu ²⁺ , bovine serum albumin, D-glucose and L-ascorbic acid is more preferably 1:4:40-320: 10~140.

The reducing agent in the present invention is preferably a co-reducing agent system of D-glucose and hydroxylamine hydrochloride, and the molar ratio of Cu ²⁺ , bovine serum albumin, D-glucose and hydroxylamine hydrochloride is more preferably 1:4:40-320:40 ~320.

The reducing agent in the present invention is preferably a co-reducing agent system of L-ascorbic acid and hydroxylamine hydrochloride, and the molar ratio of Cu ²⁺ , bovine serum albumin, L-ascorbic acid and hydroxylamine hydrochloride is more preferably 1:4:10-140:40 ~320.

The fluorescent copper nano-cluster synthesized by the invention has the advantages of bright emission of fluorescence, small size, high stability, good water solubility and the like.

Description of drawings

13 pieces of accompanying drawings of the present invention,

Fig. 1 is the influence of the hydroxylamine hydrochloride of different concentrations of embodiment 1～6 on the fluorescence intensity of synthetic fluorescent copper nanocluster;

Fig. 2 is the influence of the L-ascorbic acid of embodiment 7～13 different concentrations on the fluorescence intensity of synthetic fluorescent copper nanocluster;

Fig. 3 is the influence of the D-glucose of different concentrations of embodiment 14～21 on the fluorescence intensity of synthetic fluorescent copper nanocluster;

Fig. 4 is the influence of the sodium borohydride of different concentrations of embodiment 22～26 on the fluorescence intensity of synthetic fluorescent copper nanocluster;

Fig. 5 is the effect of sodium borohydride and D-glucose co-reductant system of different concentrations in Examples 43 to 50 on the fluorescence intensity of the synthesized fluorescent copper nanoclusters;

Fig. 6 is the effect of sodium borohydride and L-ascorbic acid co-reductant system of different concentrations in Examples 51 to 58 on the fluorescence intensity of the synthesized fluorescent copper nanoclusters;

Fig. 7 is the effect of the D-glucose and L-ascorbic acid co-reductant system of different concentrations in Examples 59 to 66 on the fluorescence intensity of the synthesized fluorescent copper nanoclusters;

Fig. 8 is the effect of D-glucose and hydroxylamine hydrochloride co-reductant system of different concentrations in Examples 67 to 74 on the fluorescence intensity of the synthesized fluorescent copper nanoclusters;

Fig. 9 is the effect of the co-reductant system of L-ascorbic acid and hydroxylamine hydrochloride with different concentrations in Examples 75 to 82 on the fluorescence intensity of the synthesized fluorescent copper nanoclusters;

Fig. 10 is the UV-vis absorption spectrogram of the fluorescent copper nanocluster and bovine serum albumin synthesized in embodiment 2;

Fig. 11 is the fluorescence excitation and emission spectrograms of the fluorescent copper nanocluster synthesized in Example 2;

Figure 12a is a transmission electron microscope image of the fluorescent copper nanocluster synthesized in Example 2;

Figure 12b is the particle size distribution of 70 particles obtained by transmission electron microscope imaging of fluorescent copper nanoclusters synthesized according to Example 2;

Fig. 13 is a spectrogram showing the stability of the fluorescent copper nanoclusters synthesized in Example 2 and the newly synthesized fluorescent copper nanoclusters of Example 2 stored at 4°C in the dark for 4 months.

detailed description

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

The specific implementation manner of the present invention will be described in detail below in conjunction with the technical solutions.

Embodiment 1～6

A kind of synthetic method of fluorescent copper nanocluster, described synthetic method is that 5mL concentration is the Cu(NO ₃ ) ₂ solution of 5mM and 5mL concentration is the bovine serum albumin solution of 20mg/mL 25 ℃ and stirs 10min, then adds hydroxylamine hydrochloride, Adjust the pH to 12 with NaOH, and let it stand at 25°C for 24 hours;

See Table 1 for the volume and concentration values of hydroxylamine hydrochloride in Examples 1-6.

The volume and concentration of the hydroxylamine hydrochloride of table 1 embodiment 1～6

Examples 7-13

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is L-ascorbic acid;

See Table 2 for the volume and concentration values of L-ascorbic acid in Examples 7-13.

The volume and concentration of the L-ascorbic acid of table 2 embodiment 7～13

Examples 14-21

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is D-glucose;

See Table 3 for the volume and concentration of D-glucose in Examples 14-21.

The volume and concentration of the D-glucose of table 3 embodiment 14～21

Examples 22-26

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is sodium borohydride;

See Table 4 for the volume and concentration values of the sodium borohydride in Examples 22 to 26.

The volume and concentration of the sodium borohydride of table 4 embodiment 22～26

Examples 27-34

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is sodium citrate;

See Table 5 for the volume and concentration values of the sodium citrate in Examples 27-34.

The volume and concentration of the sodium citrate of table 5 embodiment 27～34

Examples 35-42

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is sodium borohydride and sodium citrate;

See Table 6 for the volume and concentration values of sodium borohydride and sodium citrate in Examples 35-42.

The volume and concentration of sodium borohydride and sodium citrate of table 6 embodiment 35～42

Examples 43-50

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is sodium borohydride and D-glucose;

See Table 7 for the volume and concentration values of sodium borohydride and D-glucose in Examples 43-50.

The volume and concentration of sodium borohydride and D-glucose of table 7 embodiment 43～50

Examples 51-58

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is sodium borohydride and L-ascorbic acid;

See Table 8 for the volume and concentration values of sodium borohydride and L-ascorbic acid in Examples 51-58.

The volume and the concentration of sodium borohydride and L-ascorbic acid of table 8 embodiment 51～58

Examples 59-66

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is D-glucose and L-ascorbic acid;

The volumes and concentrations of D-glucose and L-ascorbic acid in Examples 59-66 are shown in Table 9.

The volume and concentration of D-glucose and L-ascorbic acid of table 9 embodiment 59～66

Examples 67-74

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is D-glucose and hydroxylamine hydrochloride;

See Table 10 for the volume and concentration values of D-glucose and hydroxylamine hydrochloride in Examples 67-74.

The volume and concentration of the D-glucose and hydroxylamine hydrochloride of table 10 embodiment 67～74

Examples 75-82

A method for synthesizing fluorescent copper nanoclusters, the difference from Example 1 is that the reducing agent is L-ascorbic acid and hydroxylamine hydrochloride;

See Table 11 for the volume and concentration values of L-ascorbic acid and hydroxylamine hydrochloride in Examples 75-82.

The volume and concentration of L-ascorbic acid and hydroxylamine hydrochloride of table 11 embodiment 75～82

Effect Example 1

Test the UV-vis absorption spectrum of the fluorescent copper nanocluster synthesized in Example 2 and bovine serum albumin, the results are shown in Figure 10, a is the UV-vis absorption spectrum of the fluorescent copper nanocluster synthesized in Example 2, and b is bovine serum The UV-vis absorption spectrogram of protein is obtained by Fig. 10. Compared with the obvious absorption peak of bovine serum albumin at 280nm, the fluorescent copper nanocluster synthesized in Example 2 has no obvious absorption peak in the entire UV-vis light region, The tiny hump around 280nm is due to unreacted bovine serum albumin. In addition, there is no obvious surface plasmon resonance absorption band at 560-600nm, which proves that no large-sized copper nanoparticles are formed.

Effect example 2

The fluorescent copper nanoclusters synthesized in Example 2 are tested for fluorescence excitation and emission spectra, the results are shown in Figure 11, a is the fluorescence excitation spectrum, b is the fluorescence emission spectrum, obtained from Figure 11, the Stokes shift is 260nm, which proves that the emission spectrum can be effectively Avoid interference from excitation light sources. Compared with organic dyes, the large-scale Stokes shift can effectively avoid the crossover between excitation and emission spectra.

Effect example 3

The fluorescent copper nanoclusters synthesized in Example 2 were characterized by transmission electron microscopy for their morphology and particle size. The results are shown in Figure 12a and Figure 12b. From Figure 12a and Figure 12b, the fluorescent copper nanoclusters synthesized in Example 2 are circular And evenly distributed, due to the steric protection of bovine serum albumin, the formation of large-sized metal nanoparticles and aggregates was not observed, and the particle size distribution of 70 particles was statistically analyzed and calculated to obtain the fluorescent copper synthesized in Example 2 The average size of the nanoclusters is 2.47 nm.

Effect Example 4

The fluorescent copper nanoclusters synthesized in Example 2 stored at 4°C in the dark for 4 months were compared with the newly synthesized fluorescent copper nanoclusters of Example 2. The results are shown in Figure 13, a is the newly synthesized fluorescent copper nanoclusters of Example 2. The fluorescence emission spectrum of the cluster, b is the fluorescence emission spectrum of the fluorescent copper nanocluster synthesized in Example 2 stored in the dark at 4°C for 4 months, as shown in Figure 13, although the maximum emission wavelength is red-shifted from 615nm to 649nm, but, There is no change in the fluorescence intensity, which proves that the fluorescent copper nanoclusters synthesized in Example 2 have high stability.

Claims (4)

1. A synthetic method for fluorescent copper nanoclusters, characterized in that: the synthetic method is to contain Cu ^{2 +} solution mixed with bovine serum albumin solution, then add reducing agent, adjust pH to 10～13, leave standstill Reaction 6～30h; The molar ratio of Cu ²⁺ , bovine serum albumin and reducing agent is 1:4:1-320; The reducing agent is at least one of hydroxylamine hydrochloride, L-ascorbic acid, D-glucose, sodium borohydride and sodium citrate. 2. The synthetic method according to claim 1: it is characterized in that: the reducing agent is one of hydroxylamine hydrochloride, L-ascorbic acid, D-glucose, sodium borohydride and sodium citrate. 3. synthetic method according to claim 1: it is characterized in that: described reducing agent is sodium borohydride and sodium citrate co-reductant system or sodium borohydride and D-glucose co-reductant system or sodium borohydride and L-ascorbic acid co-reductant system, or D-glucose and L-ascorbic acid co-reductant system, or D-glucose and hydroxylamine hydrochloride co-reductant system, or L-ascorbic acid and hydroxylamine hydrochloride co-reductant system. 4. The synthesis method according to claim 1: it is characterized in that: the reaction temperature is 20-40°C.